A retrospective analysis of a commercially available platelet-rich plasma kit during clinical use.

INTRODUCTION: Point-of-care analyses of platelet-rich plasma (PRP) are not routine in the orthopedic regenerative medicine field. Therefore, many physicians rely on the manufacturer's reported content for commercial preparation kits. This contributes to a knowledge gap between injectate content and patient outcome. OBJECTIVE: To assess whether the EmCyte PurePRP II 60-mL preparation kit returns PRP content that meets the manufacturer's expectations when used during routine clinical care for a heterogenous patient population, and to determine whether a change in PRP yield volume affects injectate content. Protocol A (exclusion of granulocytes and low hematocrit) and Protocol B (inclusion of granulocytes and higher hematocrit) were evaluated. DESIGN: Retrospective review. SETTING: Private practice. PARTICIPANTS: One hundred five patients (118 preparations) treated for orthopedic conditions over an 8-month period via PRP injection. Thirteen patients had two independently made preparations on different treatment days that qualified for analysis. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Complete blood count (absolute counts and calculated fold enrichment from baseline of platelets, white blood cells, red blood cells, granulocytes, lymphocytes, monocytes; and hematocrit levels). Confounding variables included age, gender, and preparation yield volume. RESULTS: During routine clinical use, the cellular content of both Protocols A and B met or exceeded the manufacturer's expectations of platelet enrichment and granulocyte inclusion or exclusion. Hematocrit values were slightly higher than anticipated from Protocol A preparations. The modification of yield volume from 7 to 4 mL led to a significant difference in platelet enrichment without affecting absolute cell counts (2.88; 95% confidence interval [CI] 1, 4.76; P = .003). Both gender and age moderately affected the level of platelet enrichment from baseline but did not significantly affect absolute platelet counts. CONCLUSION: In the absence of widespread characterization, confirming the variation in commercial PRP kits during clinical use is crucial.

Optimizing the safety of intradiscal platelet-rich plasma: an in vitro study with Cutibacterium acnes.

Aim: The most common risk associated with intradiscal injection of platelet-rich plasma (PRP) is discitis with Cutibacterium acnes. It is hypothesized that antimicrobial activity of PRP can be enhanced through inclusion of leukocytes or antibiotics in the injectate. Materials & methods: Multiple PRP preparations of varying platelet and leukocyte counts were co-cultured with C. acnes with or without cefazolin, with viable bacterial colony counts being recovered at 0, 4, 24 and 48 hours post-inoculation. Results: A direct correlation between C. acnes recovery and granulocyte counts were observed. Conclusion: We observed the greatest antimicrobial activity with the leukocyte-rich, high platelet PRP preparation combined with an antibiotic in the injectate. However, cefazolin did not completely clear the bacteria in this assay.

Bacterial toxin YafQ is an endoribonuclease that associates with the ribosome and blocks translation elongation through sequence-specific and frame-dependent mRNA cleavage.

Toxin-antitoxin (TA) systems on the chromosomes of free-living bacteria appear to facilitate cell survival during intervals of stress by inducing a state of reversible growth arrest. However, upon prolonged stress, TA toxin action leads to cell death. They have been implicated in several clinically important phenomena--bacterial persistence during antibiotic treatment, biofilm formation and bacterial pathogenesis--and serve as attractive new antibiotic targets for pathogens. We determined the mode of action of the YafQ toxin of the DinJ-YafQ TA system. YafQ expression resulted in inhibition of translation, but not transcription or replication. Purified YafQ exhibited robust ribonuclease activity in vitro that was specifically blocked by the addition of DinJ. However, YafQ associated with ribosomes in vivo and facilitated rapid mRNA degradation near the 5' end via cleavage at AAA lysine codons followed by a G or A. YafQ(H87Q) mutants lost toxicity and cleavage activity but retained ribosome association. Finally, LexA bound to the dinJ-yafQ palindrome and triggered module transcription after DNA damage. YafQ function is distinct from other TA toxins: it associates with the ribosome through the 50S subunit and mediates sequence-specific and frame-dependent mRNA cleavage at (5')AAA-G/A(3') sequences leading to rapid decay possibly facilitated by the mRNA degradosome.

Loss of the Rpb4/Rpb7 subcomplex in a mutant form of the Rpb6 subunit shared by RNA polymerases I, II, and III.

We have identified a conditional mutation in the shared Rpb6 subunit, assembled in RNA polymerases I, II, and III, that illuminated a new role that is independent of its assembly function. RNA polymerase II and III activities were significantly reduced in mutant cells before and after the shift to nonpermissive temperature. In contrast, RNA polymerase I was marginally affected. Although the Rpb6 mutant strain contained two mutations (P75S and Q100R), the majority of growth and transcription defects originated from substitution of an amino acid nearly identical in all eukaryotic counterparts as well as bacterial omega subunits (Q100R). Purification of mutant RNA polymerase II revealed that two subunits, Rpb4 and Rpb7, are selectively lost in mutant cells. Rpb4 and Rpb7 are present at substoichiometric levels, form a dissociable subcomplex, are required for RNA polymerase II activity at high temperatures, and have been implicated in the regulation of enzyme activity. Interaction experiments support a direct association between the Rpb6 and Rpb4 subunits, indicating that Rpb6 is one point of contact between the Rpb4/Rpb7 subcomplex and RNA polymerase II. The association of Rpb4/Rpb7 with Rpb6 suggests that analogous subunits of each RNA polymerase impart class-specific functions through a conserved core subunit.